We have been actively engaged in defining the molecular mechanism by which retroviral Env glycoproteins are incorporated into virus particles during the assembly process. A complete understanding of this process has been stymied by a lack of structural information about the matrix domain of Gag (MA) and the cytoplasmic tail (CT) of gp41 in virions, cell-type-specific differences in the requirement for the gp41 CT in Env incorporation, clear differences in the roles of the gp41 CT between HIV-1 and the SIVmac strain of simian immunodeficiency virus in human vs. monkey cells, and the plethora of trafficking and signaling motifs present in the CTs of retroviral Env proteins. Recently, we have made significant progress in understanding the structural requirements for Env incorporation from the perspective of MA, and will build on these advances to elucidate the role of the gp41 CT in Env incorporation. _____Several lines of evidence suggest that HIV 1 Env glycoproteins are recruited into virions via direct interactions between Env and MA; for example, mutations in both MA and the gp41 CT can block HIV 1 Env incorporation. Our recent findings strongly support the hypothesis that trimerization of the MA domain plays an important role in Env recruitment: a mutation at the putative MA trimer interface is able to rescue the Env-incorporation defect imposed by a large panel of MA mutations and a small deletion in the gp41 CT, and mutations that disrupt MA trimer formation block Env incorporation. In this project, we aim to further elucidate the structural requirements for Env incorporation, focusing first on HIV-1 and then extending our analysis to other lentiviruses and, more broadly, other retroviruses. _____We showed a number of years ago that HIV-1 Env is likely to interact, in a cell-type-dependent manner, with host cell factors that promote Env incorporation. More recent studies suggested that Env incorporation is mediated by interactions between MA and the host factor tail-interacting protein of 47 kDa (TIP47). As part of our ongoing efforts to understand the host cell machinery required for HIV-1 Env incorporation, we reevaluated the role of TIP47 in this process. A direct interaction between MA and TIP47 was confirmed by NMR spectroscopy titration experiments and surface plasmon resonance [performed in the labs of our collaborators Drs. Michael Summers (University of Maryland) and Simon Cocklin (Drexel University)]. However, in HeLa cells, TIP47 overexpression or RNAi-mediated depletion had no significant effect on HIV-1 Env incorporation, virus release, or particle infectivity. Similarly, depletion of TIP47 in the Jurkat T-cell line did not impair HIV-1 Env incorporation, virus release, infectivity, or replication. Our results thus do not support a role for TIP47 in HIV-1 Env incorporation or virion infectivity._____More recently, the Spearman lab demonstrated that another host protein, Rab11-FIP1c, plays an important role in Env trafficking and incorporation into virions. The retromer complex was also suggested to function in Env trafficking. An intriguing aspect of the cell-type-specific nature of lentiviral Env incorporation is that while in most relevant human cell types truncation of the gp41 CT blocks HIV-1 replication, SIVmac acquires gp41 CT stop codons when propagated in human cells. These stop codons revert to the wild-type sequence when the mutant viruses are propagated in monkey cells (e.g., rhesus PBMCs). Thus, the HIV-1 gp41 CT plays a positive role in virus replication, whereas the SIVmac gp41 CT plays a negative role in human cells but a positive role in monkey cells. Understanding the basis for these observations is likely to provide novel insights into the role of gp41 in lentiviral biology. We will evaluate the role of host factors in primate lentiviral Env glycoprotein incorporation and the determinants in MA and gp41 required for Env incorporation._____Although a trimeric MA crystal structure has been available since 1996, evidence for functional MA trimers has been elusive. The mechanism of HIV-1 Env recruitment into virions likewise has been unclear. We identified a point mutation in MA (62QR) that rescues the Env-incorporation defects imposed by an extensive panel of MA and Env mutations. Mapping the mutations onto the putative MA trimer reveals that the incorporation-defective mutations cluster at the tips of the trimer, at the perimeter of a putative gap in the MA lattice into which the gp41 CT could insert. By contrast, the rescue mutation is located at the trimer interface, suggesting that it confers rescue of Env incorporation via modification of MA trimer interactions. These data strongly support the existence of MA trimers in the immature Gag lattice and demonstrate that rescue of Env-incorporation defects is mediated by modified interactions at the MA trimer interface. The importance of the trimer interface in rescuing HIV-1 Env incorporation suggests that the trimeric arrangement of MA plays a critical role in permitting incorporation of Env into the Gag lattice. Inhibitors could be developed to block HIV-1 Env incorporation by disrupting this essential structural element in MA trimerization. Future work could also yield strategies to block HIV-1 Env incorporation by disrupting the function of host factors, or the interactions between host factors and either Env or Gag._____[Corresponds to Freed Project 2 in the July 2016 site visit report of the HIV Dynamics and Replication Program]